Recombinant production allows the large scale manufacturing of therapeutic proteins while avoiding the many of the difficulties and hazards of protein purification from natural sources. In the case of human proteins, recombinant manufacturing is frequently the only practical method for producing the amounts of protein required for commercial sales of therapeutic products. Recombinant production also eliminates worker exposure to human fluid and tissues, avoiding potential exposure to infectious agents such as viruses.
Recombinant manufacturing involves the expression of a DNA construct encoding for the desired protein in a recombinant host cell. The host cell can be either prokaryotic (e.g., bacteria such as Escherichia coli) or eukaryotic (e.g., yeast or a mammalian cell line). For large scale recombinant manufacturing, bacterial or yeast host cells are most commonly used, due to the ease of manipulation and growth of these organisms and also because these organisms require relatively simple growth media.
Recombinant manufacturing does, however, have its difficulties. Expression constructs must be optimized for the particular protein and for the host cell. Expression of the recombinant protein in the host cell exposes the recombinant protein to a new set of host cell enzymes, such as proteases, which can modify or even degrade the recombinant protein. Modification and degradation of the recombinant protein is, of course, undesirable, as it decreases yields and can complicate the purification of the recombinant protein.
Protamine sulfate is a compound which is well known in the art of recombinant protein production for use in removing undesired DNA from a solution. When protamine sulfate is used to remove undesired DNA, it is added to clarified lysates (i.e., the solution resulting from lysis of the host cells and the removal of cellular debris) at concentrations of about 0.1 to 0.2% (w/v). Addition of protamine sulfate causes the precipitation of DNA, allowing for easy removal by filtration or centrifugation. Addition of protamine sulfate prior to the clarified lysate stage is normally considered undesirable; if protamine sulfate is added prior to the clarified lysate stage, the resulting precipitated DNA can complicate the clarification process (for proteins accumulating in soluble form) or contaminate the "inclusion bodies" (for proteins that accumulate in insoluble form).
Sodium dodecyl sulfate (SDS) is a sulfated detergent that is occasionally used to lyse the membranes of recombinant host cells, particularly for the isolation of large plasmids. The use of SDS in lysing host cells for the recovery of recombinant protein is, however, generally avoided as SDS binds to proteins (normally denaturing them) and can be very difficult to remove.
A number of compounds are available that will increase recombinant protein yields by inhibiting host cell protease activity. Unfortunately, these compounds can be toxic (e.g., phenylmethylsulfonylfluoride(PMSF)), and are, therefore, undesirable for inclusion in a process for manufacturing a product for administration to humans or other living organisms. Other protease inhibitors are available that are not toxic, such as tripeptides, but these compounds are generally so expensive as to render them impractical for inclusion in a commercial manufacturing process.
Accordingly, there is a need in the art for methods that increase the yield of recombinant proteins without the use of toxic or prohibitively expensive compounds.